Download AgCaspar depletion regulated immune genes with diverse

Supplementary Text
Primers for RNAi assays:
CactusF: 5’-TAATACGACTCACTATAGTAACACTGCGCTTCATTTGG-3’
CactusR: 5’-TAATACGACTCACTATAGGCCCTTTTCAATGCTGATGT-3’
Caspar F: 5’-TAATACGACTCACTATAGCCGCTTTTCTAAACGCTGTC-3’
CasparR: 5’-TAATACGACTCACTATAGAAACAGGTTGCATGTGTGGA-3’
Rel1F: 5′- TAATACGACTCACTATAGATCAACAGCACGACGATGAG-3′
Rel1R: 5′- TAATACGACTCACTATAGTCGAAAAAGCGCACCTTAAT -3′
Rel2F: 5′- TAATACGACTCACTATAGCGGAGAAGTCGAAGAAAACG-3′
Rel2R: 5′-TAATACGACTCACTATAGCACAGGCACACCTGATTGAG -3′
AnAlbCasparF: 5’: TAATACGACTCACTATAGGCGAAGCGACAGAAGGAG-3’
AnAlbCasparR:
TAATACGACTCACTATAGTCTTAAGTCTCTGCGAGGCCAGCTTGAGAT-3’
AnAlbCactusF: 5’: TAATACGACTCACTATAGCTGAACATTCAGAACGAT-3’
AnAlbCactusR: 5’: TAATACGACTCACTATAGCGGAGATTTTCCTTCCCT-3’
FBN9F: 5′- TAATACGACTCACTATAGCCAAGATGTCGGGCAAGTAT -3′
FBN9R: 5′- TAATACGACTCACTATAGTTGTGGTACGTCAGCGAGTC -3′
TEP1F: 5′- TAATACGACTCACTATAGGTTTGTGGGCCTTAAAGCGCTG -3′
TEP1R: 5′- TAATACGACTCACTATAGGACCACGTAACCGCTCGGTAAG -3′
LRRD7F: 5′- TAATACGACTCACTATAGTCGGTGAGCAACAGTTTGAC -3′
LRRD7R: 5′- TAATACGACTCACTATAGCTTCATTCCCGCTAATGCTC-3′
5’:
Underlined sequence corresponds to T7 promoter sequence. AnAlb primers refer to RNAi
primers used specifically for A. albimanus and A. stephensi.
Primers for qRT-PCR:
CasparVerifyF: Same as RNAi Forward Primer
CasparVerifyR: 5’-GAACGGCTGCGCTTTAACA-3’
CactusVerifyF: Same as RNAi Forward Primer
Cactus VerifyR: 5’-TCGTTCAAGTTCTGTGCAAGTGT-3’
Cecropin1F: 5’-AGACCAACCAACCACCAAAC-3’
Cecropin1R: 5’-GTTAGCAGAGCCGTCGTCTT-3’
Gambicin1F: 5’-TGCGAGATGTAAAAGCATCG-3’
Gambicin1R: 5’-CCAACGTCTGGCACTGATTA-3’
Defensin1F: 5’-GCGGTTCCAAAGTTCCGACA-3’
Defensin1R: 5’-AGCGGGACACAAAATTGTTC-3’
CLIPA9F: 5’-GTTGGGGACTGGACGTTAGA-3’
CLIPA9R: 5’- GTACCCTTCGATCGTGTCGT-3’
CathepsinD: 5’- TTCTCGGATATGCCGATTTC-3’
CathepsinD: 5’- TCTCGAACGAGGACGACTTT-3’
Verify Primers were used to verify silencing efficiency.
validated previously [1, 6].
Other silencing treatments were
Supplementary Figure and Table Legends
Table S1: Gene expression following single or double knock down of negative regulators and
Rel factors Column 1: Gene name; Column 2: Array-derived gene expression values in cactusor caspar-silenced mosquitoes; Column 3: Real-time PCR-derived expression values in cactusor caspar-silenced mosquitoes; Column 4: Real time PCR-derived gene expression values in
cactus/rel1 or caspar/rel2 double silenced mosquitoes Numbers represent ratio of silenced to
control mosquitoes All values represent the average of 3 biological replicas A) cactus-silenced
groups B) caspar-silenced groups
Table S2: Microarray-derived gene expression values for immune genes.
Column 1 indicates gene name, Column 2 indicates the Ensembl transcript ID for each gene and
Column 3 indicates the log transformed fold change in gene expression due to: A) cactus
silencing or B) caspar silencing
Table S3: cactus gene silenced transcriptome expressed as log2 transformed cactus gene
silenced/GFP dsRNA expression ratio.
Table S4: caspar gene silenced transcriptome expressed as log2 transformed caspar gene
silenced/GFP dsRNA expression ratio.
Analysis of individual genes regulated by Cactus and Caspar depletion
Caspar depletion regulated immune genes with diverse functions, such as those encoding the
Clip domain serine protease CLIPB17, the serine protease inhibitor serpin5, pro-phenoloxidase1,
several fibrinogen immuno-lectins (FBNs), two scavenger receptors (SCRs) and two
peroxidases, as well as several genes involved in apoptosis, were regulated by knockdown of
cactus but not caspar. Genes up-regulated following caspar but not cactus silencing encode a
serine protease (CLIPB4), a putative homolog of mammalian MD-2 (AgMDL8), SCRB1 and an
FBN yet down-regulated a serine protease and FBN9. The 27 genes that were up-regulated upon
the depletion of either factor included 8 immune genes: 3 AMPs (Cec1, Cec3 and Def1), 3 serine
proteases (CLIPA9, CLIPD4 and one novel), TEP3 and FBN37. Both treatments repressed two
genes, one of which is related to immunity. Only one gene displayed the opposite pattern of
regulation; the anti-Plasmodium factor LRRD7 (leucine rich repeat domain 7, also known as
APL2) showed enrichment upon cactus silencing and repression upon caspar silencing (Figure 3
and Table S1).
Cactus depletion specifically induced transcription of the genes for CLIPB17 serine
protease, which is known to promote parasite melanization and SRPN5, a serine protease
inhibitor, yet down-regulates that of phenoloxidase enzyme PPO1 [46]. This signature is likely
to represent a regulatory mechanism for melanization-dependent defenses directed by the Toll
pathway. Genes encoding three programmed cell death proteins, Bax inhibitor, PCD4 and
CathepsinD, were also influenced by cactus silencing which may relate to the suggested role of
Toll-like receptors as sensors for autophagy in mammals though the signaling for insect
autophagy is as yet unknown [47]. Two peroxidases are increased upon cactus silencing, one of
which is up-regulated in the A. gambiae midgut during P. berghei infection, possibly as part of
the apoptotic response of midgut epithelial cells [48].
caspar silencing influenced transcription of significantly fewer immune genes, which is a
likely indication that this negative regulator affects a certain branch of the Imd pathway: the
Dredd-dependent activation of Rel2 [16, 49]. This selectivity, together with our use of adult
mosquitoes (containing a variety of immune and non-immune tissues) instead of cell lines and a
timepoint reflecting late expression, were most likely the reasons for the absence of detectable
differential expression of some previously reported target genes of the Imd pathway, such as
PGRP-LC and LRIM1, following Caspar depletion. Enhanced expression was, however,
observed for the antimicrobial peptide genes cec1, cec3 and cef1, which are targets of the Imd
pathway and may exhibit specificity with regard to the Rel isoform to which they respond [4,
28]. We also note that the expression of the anti-microbial peptide gambicin (gam1) following
silencing of either cactus or caspar did not pass the induction threshold we used for array
analysis but was picked up as weakly induced by the sensitivity of real time PCR.
The association of a highly potent anti-Plasmodium activity with Caspar depletion
suggests activation of Rel2 results in transcriptional activation of anti-Plasmodium effectors. A
limited number of such effectors that are controlled by the Rel1 and Rel2 factors have already
been identified [4-6]. Caspar depletion would therefore be expected to cause transcriptional
activation of genes responsible for this dramatically refractory phenotype and, similarly, Cactus
depletion would be expected to induce genes mediating its observed infection phenotype. By
specifically targeting these molecules that are unique to one Imd branch or the other, we can
begin to understand the complexity of the Imd pathway and it’s regulation of anti-Plasmodium
defense (see main text).
The overlaps between Rel1- and Rel2-induced gene expression profiles were extensive,
including several innate immune genes (Figure 3B). Three anti-microbial peptides, cec1, cec3
and def1 were up-regulated by both Cactus and Caspar RNAi-mediated depletion. This result is
not surprising, since dual regulation of anti-microbial peptides by both the Toll and Imd
pathways has been shown in Drosophila and mosquito cell lines [16, 50, 51]. Four serine
proteases, including CLIPA9 and CLIPD4, showed enrichment in response to the activation of
both Rel factors. Serine proteases, such as those with Clip domains, are responsible for the
signal amplification that occurs between pathogen recognition events and the activation of the
Toll or Imd pathways and are also linked to the melanization of parasites and other foreign
bodies [46, 52, 53].
Both Cactus- and Caspar-depleted cohorts showed elevated expression of the TEP3 gene,
a member of the thioester-containing protein family that also includes TEP1, a potent antiPlasmodium molecule, has been reported to be up-regulated during bacteria and malaria
infection [3, 54]. FBN37, a putative pattern recognition receptor, is another immune gene that
was enriched in both gene expression profiles. The enrichment of transcripts encoding the
frizzled protein in both Cactus- and Caspar-depleted mosquitoes is intriguing, since frizzled has
been implicated as a key mediator of melanization and the actin polymerization that occurs
around the rodent P. berghei parasites in the mosquito midgut, and may be linked to TEP1
activity [55]. One anti-Plasmodium factor, LRRD7 (APL2), was induced by cactus silencing but
repressed by caspar silencing (see main text for further analysis). LRRD7 (APL2) is of
particular significance because this gene is located in a region conferring resistance to
Plasmodium, and silencing of this gene causes P. falciparum infection to increase by two-fold
(see main text for further analysis) [6, 32].
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